Evaluation of Anti-Inflammatory Activity and In-vitro Antioxidant Activity of Indian Mistletoe, the Hemiparasite Dendrophthoe falcate L. F. (Loranthaceae)

Methanolic and aqueous extracts of Dendrophthoe falcata Linn. leaves which belongs to the Loranthaceae family, were evaluated through DPPH (1, 1-diphenyl -2-picryl-hydrazyl), antilipid peroxidation and nitric oxide scavenging methods to assess the antioxidant activity. Methanolic and aqueous extracts of Dendrophthoe falcata leaves were also evaluated for their anti-inflammatory activity by carrageenan and cotton pellet induced granuloma tests for their effect on the acute and chronic phase inflammation models in rats. It was found that the methanolic extract of Dendrophthoe falcata leaves demonstrates potent antioxidant activity as compared to aqueous extraction of Dendrophthoe falcata leaves for DPPH (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging, anti-lipid peroxidation and nitric oxide scavenging activity respectively (having IC50 value 77.8, 79.36 and 86.2, 144, 87, 104). The maximum inhibition for aqueous extract of Dendrophthoe falcata leaves (30.95%) and methanolic extract of Dendrophthoe falcata leaves (23.41%) were obtained at a dose of 300 mg/Kg after 4h of drug treatment in carrageenan induced paw edema, whereas diclofenac sodium (standard drug) produced 42.85% inhibition. In the chronic model (cotton pellet induced granuloma), aqueous extracts of Dendrophthoe falcata leaves and methanolic extracts of Dendrophthoe falcata leaves (at doses of 300 mg/Kg), phenylbutazone as standard drug showed decreased formation of granuloma tissue by 51%, 48%, 53% respectively. In addition, the total phenolic and flavonoid content of aqueous extracts of Dendrophthoe falcata leaves and methanolic extracts of Dendrophthoe falcata leaves were found to be 2.12 % w/w, 4.39 % w/w, 0.31 mg/g and 0.85 mg/g respectively. Thus the results indicate that methanolic and aqueous extracts of Dendrophthoe falcataleaves on animal models have potent anti-inflammatory and in-vitro antioxidant effects.


Introduction
The genus Dendrophthoe is evergreen, shrubby, partial parasites, distributed in the tropical and sub-tropical regions of the old world. The whole parasitic plant Dendrophthoe falcata is used in indigenous system of medicine as cooling, bitter, astringent, aphrodisiac, narcotic and diuretic and is also useful in pulmonary tuberculosis, asthma, menstrual disorders, swelling wounds, ulcers, renal and vesicle calculi and vitiated conditions of kapha and pitta. The decoction of Dendrophthoe falcata is used by women as antifertility agent. The Dendrophthoe falcata also have anticancer activity (1). Dendrophthoe falcate that is branched angiospermic hemiparasite, was most frequently observed on hosts Mangifera indica (Anacardiaceae), Melia azadirecta (Meliaceae) and Psidium guayava (Myrtaceae). Barks of Dendrophthoe falcata are grey, its leaves are thick, coriaceous, much variable in shape usually are stout, unilateral racemes often two from an concave and scarlet or orange or less commonly pink or white in colour. Anthers are linear, equal Berries of Dendrophthoe falcata are 8-13 mm long ovoid oblong, pink, smooth crowned by a cup-shaped calyx (2). The genus Dendrophthoe comprises of 20 species and about 7 species are found in India. Members of genus Dendrophthoe are reported to have anti-oxidant, anti-microbial, anticancer, antidiabetic (3), anti-lithiatic and antihypertensive activity (4).
The present study was undertaken to investigate the in-vitro antioxidant and anti-Dendrophthoe falcata leaves.

Materials
The leaves of Dendrophthoe falcata parasitic on Mangifera indica (Anacardiaceae) were collected from western ghat region of maharashtra (India) in February 2005. The Dendrophthoe falcata plant specimen was authenticated from botanical survey of india, pune (Voucher specimen no. PSH-1). The airdried leaves of D. falcata were pulverized and the powdered material was extracted with methanol (80 %) and chloroform water by cold maceration at room temperature for seven days, chloroform water is used to avoid the fungal growth during of distilled water). The methanolic and aqueous extracts of Dendrophthoe falcata leaves were concentrated on a rotary vacuum evaporator at reduced pressure, which gave a yield (4.16 and 9.42 % w/w). The proximate phytochemical analysis of methanol and aqueous extracts of Dendrophthoe falcata leaves shows presence of

Animals
Wistar rats, of either sex, weighing 180-250 g were used. They were housed under standard and dark-light cycle. They were given standard diet and water ad libitum. All the animals were carefully monitored and maintained in accordance with CPCSEA guidelines on control and supervision of experimental animals for 15 days. The ethical clearance was obtained from the Institutional Animal Ethics Committee (Approval no.651/02/c/CPCSEA) before the experiment.

Carrageenan-induced rat paw edema
using Carrageenan-induced rat paw edema according to method described by Winter et al. (8). The animals were starved overnight before the experiment to ensure uniform hydration. Fasting rats were divided into eight groups each carrying six animals. Group I served as a control orally. Diclofenac sodium (5 mg/Kg, p.o.) was administered to group II as standard. Groups III-VIII received the aqueous and methanolic extracts of Dendrophthoe falcata leaves at doses of 100, 200 and 300 mg/Kg as an aqueous of 1% w/v carrageenan suspension was injected subcutaneously into plantar surface of the right hind paw. The paw volume was measured using the plethysmometer at 60, 120, 180 and 240 min after carrageenan injection.

Effects in cotton pellet granuloma
The rats were divided into eight groups and each group consisted of six animals. After shaving the fur, the animals were anaesthetized using ketamine. Sterile pre-weighed cotton pellets (20 ± 1 mg) were implanted in the auxiliary region of each rat through a single needle incision. Control standard (phenylbutazone, 150 mg/Kg, p.o.), aqueous extracts of Dendrophthoe falcata leaves (100, 200, 300 mg/Kg) and DFM (100, 200, 300 mg/Kg) were administered to the respective group of animals for seven consecutive days from the day of cotton pellet implantation. On the eighth day, animals were anaesthetized again; the cotton pellets were removed surgically and made free from extraneous tissues. The pellets in the dry weight of the pellets was regarded as a measure of granuloma formation (9).

Anti-lipid peroxidation activity Anti-lipid peroxidation in liver homogenate
Preparation of liver homogenate: The liver was perfused with ice cold 0.15 M KCl via portal vein. The perfused liver was isolated and 10% (w/v) homogenate was prepared using a tissue The homogenate was used to study in-vitro lipid peroxidation (11).
concentration of drug extracts, was prepared. incubation, the reaction was stopped by adding hydroxyl toluene (BHT). These reaction mixtures were heated for 60 min at 80 º C. They were also cooled and centrifuged at 5000 rpm for 15 min. The absorbance of the supernatant was measured at 532 nm against a blank which contained all reagents except liver homogenate and drug. Identical experiments were performed to determine the normal (without drug and ferric chloride) and induced (without drug) lipid peroxidation.

Estimation of total phenolics and total
The total phenolic of extracts was determined using folin-ciocalteu reagent (13). The extracts and the absorbance at 760 nm was measured 2 determined through the reported method (14). In brief, the extract was diluted with 80% of extract was added to test tube containing 0.1 ethanol. After 40 min at room temperature, the absorbance was determined at 415 nm with was calculated according to a standard curve established with quercetin as reference. Quercetin and folin-ciocalteu reagent were obtained from Sigma-Aldrich, Germany.

Statistical analysis
The results are presented as Mean ± SEM oneway analysis of variance (ANOVA) followed by Dunnett's t-test for multiple comparisons, was used for statistical evaluation. The p-values less

Results
The intraplantar injection of carrageenan in the hind paw induced gradual increase in the edema paw volume in the control group. Aqueous extracts of Dendrophthoe falcata leaves and methanolic extract of Dendrophthoe falcata leaves at doses of 100, 200 and 300 edema formation in rat paw, 240 min after the carrageenan challenge (Table 1). The standard drug, Diclofenac sodium at dose 5 mg/Kg, marked reduction in paw edema. Aqueous extracts of Dendrophthoe falcata leaves and methanolic extract of Dendrophthoe falcata leaves at doses 0.01) inhibited granuloma formation (Table 2). Phenylbutazone (150 mg/Kg p.o.) -a standard

(min) 6(min) 12(min) 18(min) 24(min)
1% : p < 0.01 * : p < 0.05 Values are expressed as Mean ± SEM, one-way analysis of variance followed by Dunnette's multiple comparison t-tests. an experimental animal model for evaluation of (8) and is believed to be biphasic. The initial phase is due to the release of histamine, serotonin of carrageenan. The more pronounced second phase is attributed to release of bradykinin and prostaglandin. The cotton pellet granuloma bioassay is considered as a model for studies as a typical feature of established chronic (16). The aqueous extracts of Dendrophthoe falcata leaves and methanolic extract of Dendrophthoe falcata leaves exhibited rats in the cotton pellet-induced granuloma. This means that aqueous extracts and methanolic extract of Dendrophthoe falcata leaves may be result of the present study indicates that the crude fraction of aqueous and the methanol extracts of Dendrophthoe falcate Oxidative stress is major upstream in component signaling-cascade involved in molecules and chemoactractant production. Thus it will be relevant to investigate the drug-elicited marked reduction in granuloma formation. In addition, methanolic extract and aqueous extract of Dendrophthoe falcata leaves showed potent antioxidant activity in different in-vitro models like DPPH (1, 1-diphenyl -2-picryl-hydrazyl) radical scavenging, antilipid peroxidation and nitric oxide scavenging activity, having IC 50 values 77.8, 79.36 and 86.2, 144, 87, 104 mcg respectively. However, methanolic extract of Dendrophthoe falcata leaves shows good anti-oxidant activity as compared to aqueous extract of Dendrophthoe falcata leaves. In addition, the total phenolic Dendrophthoe falcata leaves and methanolic extract of Dendrophthoe falcata leaves was found to be 2.12% w/w, 4.39% w/w, 0.31 mg/g and 0.85 mg/g respectively, which plays the major role in controlling antioxidants (15).

Discussion
The aqueous extracts and methanolic extract of Dendrophthoe falcata suppressed the carrageenan induced rat paw edema 4 h after carrageenan challenge. Carrageenan induced rat paw edema is commonly used as